Ghrelin--not just another stomach hormone

Growth hormone (GH) secretagogues (GHSs) are non-natural, synthetic substances that stimulate GH secretion via a G-protein-coupled receptor called the GHS-receptor (GHS-R). The natural ligand for the GHS-R has been identified recently; it is called ghrelin. Ghrelin and its receptor show a widespread distribution in the body; the greatest expression of ghrelin is in stomach endocrine cells. Administration of exogenous ghrelin has been shown to stimulate pituitary GH secretion, appetite, body growth and fat deposition. Ghrelin was probably designed to be a major anabolic hormone. Ghrelin also exerts several other activities in the stomach. The findings that ghrelin is produced in mucosal endocrine cells of the stomach and intestine, and that ghrelin is measurable in the general circulation indicate its hormonal nature. A maximal expression of ghrelin in the stomach suggests that there is a gastrointestinal hypothalamic-pituitary axis that influences GH secretion, body growth and appetite that is responsive to nutritional and caloric intakes.     

Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice

Although ribonucleases H (RNases H) have long been implicated in DNA metabolism, they are not required for viability in prokaryotes or unicellular eukaryotes. We generated Rnaseh1(-/-) mice to investigate the role of RNase H1 in mammals and observed developmental arrest at E8.5 in null embryos. A fraction of the mainly nuclear RNase H1 was targeted to mitochondria, and its absence in embryos resulted in a significant decrease in mitochondrial DNA content, leading to apoptotic cell death. This report links RNase H1 to generation of mitochondrial DNA, providing direct support for the strand-coupled mechanism of mitochondrial DNA replication. These findings also have important implications for therapy of mitochondrial dysfunctions and drug development for the structurally related RNase H of HIV.     

Heregulin is sufficient for the promotion of tumorigenicity and metastasis of breast cancer cells in vivo

Resistance of breast carcinomas to hormonal therapy is a clinical obstacle for the treatment of breast cancer. The molecular mechanisms and the factors involved in the progression of tumors from an estrogen (E2)-dependent to an E2-independent phenotype are not entirely understood. Heregulin (HRG) is a pleiotropic growth factor that binds to the erbB family of receptors, which are correlated with breast cancer progression and an aggressive phenotype in the breast carcinomas overexpressing the receptors. Previous studies in transgenic mice have shown that HRG is sufficient to induce mammary gland transformation and proliferation in the presence of hormonal stimulation. However, these studies did not address the important issue of the E2 independence that is part of the progression of breast cancer. In this study, we investigated the role of HRG in E2 independence. We were able to determine that HRG up-regulation was sufficient for the development of mammary tumors in the absence of E2 stimulation, a situation that mimics the progression of the human disease. We demonstrated that in ovariectomized nude mice, HRG induced E2 independence and antiestrogen resistance and promoted metastasis and preneoplastic transformation of the adjacent mouse mammary tissue. We show that one of the mechanisms by which HRG achieves the aggressive phenotype may be mediated via an increase in activated mitogen-activated protein kinase, an increase in a matrix-degrading enzyme, MMP-9, and the overexpression of vascular endothelial growth factors. The up-regulation of these genes occurred in the absence of any additional stimulation, in an autocrine manner. Our data provide new insights into the mechanisms of breast cancer progression in vivo, and reinforce the important role that HRG plays in this process.     

Oral cancer,cigarette smoke and mitochondrial 18 kDa translocator protein (TSPO) — In vitro,in vivo,salivary analysis

Oral cancer features high rates of mortality and morbidity, and is in dire need for new approaches. In the present study we analyzed 18 kDa translocator protein (TSPO) expression in oral (tongue) cancer tumors by immunohistochemistry. We also assayed TSPO binding in human tongue cancer cell lines and in the cellular fraction of saliva from tongue cancer patients, heavy cigarette smokers, and non-smoking healthy people as controls. Concurrently, TSPO protein levels, cell viability, mitochondrial membrane potential (Δψ_m), and general protein levels were analyzed. TSPO expression could be significantly enhanced in oral cancer tumors, compared to unaffected adjacent tissue. We also found that five-year survival probability dropped from 65% in patients with TSPO negative tumors to 7% in patients with highly expressed TSPO (p < 0.001). TSPO binding capacity was also pronounced in the human oral cancer cell lines SCC-25 and SCC-15 (3133 ± 643 fmol/mg protein and 6956 ± 549 fmol/mg protein, respectively). Binding decreased by 56% and 72%, in the SCC-25 and SCC-15 cell lines, respectively (p < 0.05) following CS exposure in cell culture. In the cellular fraction of saliva of heavy smokers TSPO binding was lower than in non-smokers (by 53%, p < 0.05). Also the cellular fraction of saliva exposed to CS in vitro showed decreased TSPO binding compared to unexposed saliva (by 30%, p < 0.001). Interestingly, oral cancer patients also displayed significantly lower TSPO binding in the cellular fraction of saliva compared to healthy controls (by 40%, p < 0.01). Our results suggest that low TSPO binding found in the cellular fraction of saliva may depend on genetic background as well as result from exposure to CS. We suggest that this may be related to a predisposition for occurrence of oral cancer.     

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.     

Bim and Bcl-2 mutually affect the expression of the other in T cells

The life and death of T cells is controlled to a large extent by the relative amounts of Bcl-2-related proteins they contain. The antiapoptotic protein Bcl-2 and the proapoptotic protein Bim are particularly important in this process with the amount of Bcl-2 per cell dropping by about one-half when T cells prepare to die. In this study we show that Bcl-2 and Bim each control the expression of the other. Absence of Bim leads to a drop in the amount of intracellular Bcl-2 protein, while having no effect on the amounts of mRNA for Bcl-2. Conversely, high amounts of Bcl-2 per cell allow high amounts of Bim, although in this case the effect involves increases in Bim mRNA. These mutual effects occur even if Bcl-2 is induced acutely. Thus these two proteins control the expression of the other, at either the protein or mRNA level.     

The mutT defect does not elevate chromosomal fragmentation in Escherichia coli because of the surprisingly low levels of MutM/MutY-recognized DNA modifications

Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT (8-oxo-dGTPase) of Escherichia coli hydrolyze noncanonical DNA precursors to prevent incorporation of base analogs into DNA. Previous studies reported dramatic AT-->CG mutagenesis in mutT mutants, suggesting a considerable density of 8-oxo-G in DNA that should cause frequent excision and chromosomal fragmentation, irreparable in the absence of RecBCD-catalyzed repair and similar to the lethality of dut recBC and rdgB recBC double mutants. In contrast, we found mutT recBC double mutants viable with no signs of chromosomal fragmentation. Overproduction of the MutM and MutY DNA glycosylases, both acting on DNA containing 8-oxo-G, still yields no lethality in mutT recBC double mutants. Plasmid DNA, extracted from mutT mutM double mutant cells and treated with MutM in vitro, shows no increased relaxation, indicating no additional 8-oxo-G modifications. Our DeltamutT allele elevates the AT-->CG transversion rate 27,000-fold, consistent with published reports. However, the rate of AT-->CG transversions in our mutT(+) progenitor strain is some two orders of magnitude lower than in previous studies, which lowers the absolute rate of mutagenesis in DeltamutT derivatives, translating into less than four 8-oxo-G modifications per genome equivalent, which is too low to cause the expected effects. Introduction of various additional mutations in the DeltamutT strain or treatment with oxidative agents failed to increase the mutagenesis even twofold. We conclude that, in contrast to the previous studies, there is not enough 8-oxo-G in the DNA of mutT mutants to cause elevated excision repair that would trigger chromosomal fragmentation.